This research project is a direct extension of Research Project 4 in the prior granting period. Here we will use state-of-the-art genetic and genomic tools to elucidate the determinants of plasma low density lipoproteins (LDL)-cholesterol (C) levels in humans. Our ultimate goals are functional and practical: to identify genes and sequence variations that affect clinical outcomes and that provide new targets for therapeutic intervention. The most effective cholesterol-lowering agents elevate LDL receptors in the liver by either inhibiting hepatic cholesterol synthesis or blocking intestinal cholesterol absorption. A genetic analysis of these interlocking pathways will be the focus of this grant. First, we will sequence coding and conserved noncoding regions of key genes involved in dietary sterol absorption and hepatic cholesterol metabolism in selected individuals from a large (n=3,550), ethnically diverse population (The Dallas Heart Study). Candidate genes will be sequenced from individuals who have the highest (>95th percentile) and the lowest (<5th percentile) fractional absorption of cholesterol and plasma levels of LDL-C. This approach is supported by our recent findings that these individuals are enriched for nonsynonymous (NS) sequence variants that contribute to variation in plasma lipoprotein levels (1, 2). We will then test for associations between the NS sequence variants identified and plasma levels of sterols and lipoproteins in the entire Dallas Heart Study (DHS). Positive associations wilt be validated by replication in the Atherosclerosis Risk in Communities Study (ARIC). Second, we will use chip-based oligonucleotide hybridization to comprehensively assay common single nucleotide polymorphisms (SNPs) in candidate genes in the DHS. Association studies will be performed using individual SNPs and collections of linked SNPs (haplotypes) to identify alleles associated with differences in plasma sterol and LDL-C levels. Positive associations will be validated in the ARIC study. Third, we will assay 12,000 common SNPs distributed among 6,469 genes that result in an amino acid substitution. The SNPs associated with plasma levels of sterols and with LDL-C in the DHS will be validated in the ARIC study. This analysis will provide the opportunity to identify new genes contributing to inter-individual variation in plasma LDL-C levels. Fourth, we will continue to focus on individuals and families with unusual lipoprotein phenotypes to find new genes that play significant roles in cholesterol metabolism. At the forefront of these studies will be the analysis of a recently-identified family in which the proband has extreme hyper-responsiveness to dietary cholesterol. This four-pronged approach will provide an unbiased evaluation of the full spectrum of sequence variations that contribute to inter-individual differences in cholesterol absorption and plasma LDL-C levels in the general population. These approaches will be complemented by functional studies in cultured cells or in genetically-modified mice in collaboration with investigators in Research Projects 1, 2, 3, 5, and 6.